Two-dimensional electrophoretic/chromatographic separations combined with electrospray ionization FTICR mass spectrometry for high throughput proteome analysis

Abstract

A two-dimensional separation strategy combined with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) is being developed for high throughput proteomic analyses. Capillary isoelectric focusing (CIEF) coupled online with a robotic fraction collector is used to separate and collect microliter fractions of soluble Saccharomyces cerevisiae (yeast) proteins eluting from the capillary into a microtiter plate. Following tryptic digestion of each fraction, the resultant peptides are separated using capillary high performance liquid chromatography (HPLC) and analyzed by online ESI-FTICR. Protein identification is based upon the use of the experimentally measured peptide masses as accurate mass tags, augmented by conventional MS/MS methods as necessary, to identify proteins predicted from the yeast genome sequence. This new separation strategy is being evaluated using proteins extracted from yeast grown in natural isotopic abundance and ¹⁵N-enriched media. Two isotopically distinct versions of each peptide are thus observed in the ESI-FTICR spectra. The mass differences between the two versions are used to determine the number of nitrogen atoms in the peptide, and provide an additional constraint that aids protein identification. More importantly, the use of this stable-isotope labeling strategy enables the generation of “comparative displays” of the precise relative protein abundances. This two-dimensional separation strategy combined with ESI-FTICR analysis is expected to be highly amenable to automation. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 383–390, 2000